1. Field of the Invention
This invention relates to a pathogen-specific composition comprising a peptidoglycan hydrolase, LysK endolysin, and another peptidoglycan hydrolase, lysostaphin. The LysK endolysin specifically attacks the peptidoglycan cell wall of untreated, live staphylococci including S. aureus and methicillin-resistant Staphylococcus aureus (MRSA); lysostaphin is a potent anti-staphylococcal bacteriocin. The LysK endolysin—lysostaphin dual enzyme composition acts synergistically and lyses untreated, live MRSA with enhanced efficacy at dosages that are ineffective when used alone. The invention further relates to methods of treating Staphylococcus-induced diseases such as mastitis and staphylococcal infections, such as MRSA-induced infections.
2. Description of the Relevant Art
Antimicrobial resistance is such a nationwide concern that many agencies within the Department of Health and Human Services (DHHS), e.g. FDA, USDA, NIH, and CDC have formed a taskforce to address the issue Retrieved from the Internet: <URL: cdc.gov/drugresistance/actionplan/2005report/index.htm. Multi-drug resistance pathogens are no longer just nosocomiaf, but are now often community acquired (File, Jr. 2007. Cleve. Clin. J. Med. 74(Suppl. 4): S6-11 , 86-11; Furuya et al. 2007. Am. J. Infect. Control 35:359-366). Methicillin resistant Staphylococcus aureus (MRSA) is one of the most notorious drug resistant pathogens; the press has been filled with recent articles about the threat of these new “superbugs” for which new antimicrobials are sorely needed. The DHHS antimicrobial taskforce recommendations include research and development of novel antimicrobials that avoid resistance development.
Bacteriophage endolysins are peptidoglycan hydrolases that normally help the newly replicated phage particles escape from the host bacteria at the end of the phage lytic cycle. Each lytic phage genome encodes proteins that degrade the bacterial cell wall peptidoglycan and allow the newly replicated phage to escape. In addition to causing cell lysis when released from within the host, they are also evolving as a new class of candidate antimicrobial able to lyse Gram positive cells when exposed externally “from without.” They are uniquely specific to the cell wall peptidoglycan of their host (or closely related species), thus reducing the risk of resistance development in non-pathogenic commensal bacteria associated with broad range antibiotic use. In addition, presumably due to the co-evolution of phage and host of the few phage lysins that have been examined, all are refractory to resistance development, making them ideally suited to address the current problem of multi-drug resistant pathogens (reviewed in Fischetti, V. A. 2005. Trends Microbiol. 13: 491-496).
Many phage endolysins show promise in pre-clinical trials when used to cure animal models of human disease. A streptococcal bacteriophage lytic enzyme was successful in the treatment of streptococci from rats in an experimental endocarditis (Entenza et al. 2005. Antimicrobial Agents and Chemotherapy 49: 4789-4792). Similarly, phage lytic enzymes achieved mucosal clearing in streptococcal infection models when applied to the murine vagina, oropharynx (Cheng et al. 2005. Antimicrob. Agents Chemother. 49: 111-117; Fischetti, V. A. 2003. Ann. N. Y. Acad. Sci. 987: 207-214; Loeffler et al. 2003. Infection and Immunity 71:6199-6204), and oral cavity (Nelson et al., 2001. Proc. Nat. Acad. Sci. U.S.A. 98 (7): 4107-4112). Murine models of anthrax were also cured with endolysins (Schuch et al. 2002. Nature 418: 884-889). In vitro studies indicate serum antibodies to phage endolysins slowed but did not block in vitro killing of the target microbes Bacillus anthracis, Streptococcus pyogenes, or Streptococcus pneumoniae (Fischetti 2005, supra; Jado et al. 2003. J. Antimicrob. Chemother. 52 (6): 967-973; Loeffler et al., supra).
One recently described phage endolysin, LysK, can kill a wide range of staphylococci including multiple MRSA in plate lysis assays (O'flaherty et al. 2005. J. Bacteriol. 187: 7161-7164). This endolysin from the phage K is virtually identical to the phage 812 (ABL87139) and phage G1 (electronically spliced ORF 42 and 60 of genome AY954969) endolysins. Interestingly, each contains an intron in their endolysin gene, an unusual feature among phage genomes. Blast analysis of the LysK protein sequence reveals two lytic domains, a Cysteine, Histidine-dependent Amidohydrolase/Peptidase (CHAP) endopeptidase domain, an amidase (N-acetyl-muramyl-L-alanine amidase) domain, and a C-terminal SH3b cell wall binding domain (O'Flaherty et al., supra). It is common for phage endolysins to have an N-terminal lytic domain (or two) with a C-terminal cell wall binding domain (Loessner, M. J. 2005. Curr. Opin. Microbiol. 8 (4): 480-487), although recently an endolysin with two lytic domains flanking two mid-protein cell wall binding domains (Cpl-1) was reported for the LambdaSa2 prophage (Pritchard et a. 2007. Appl. Environ. Microbiol. 73: 7150-7154).
Another potent peptidoglycan hydrolase antimicrobial is lysostaphin which cleaves the pentaglycine interpeptide bridge of S. aureus cell walls. Lysostaphin was discovered more than 50 years ago (Browder et al. 1965. Biochem. BioPhys. Res. Comm. 19: 383-389), and is a potent anti-staphylococcal bacteriocin synthesized by S. simulans that kills S. aureus through digestion of the peptidoglycan with high species-specificity. This specificity was first shown to require the C-terminal 92 amino acids, with this protein fragment able to direct binding of heterologous proteins to the S. aureus cell wall (Baba and Schneewind. 1996. EMBO Journal 15: 4789-4797). The exact site of binding was demonstrated by two laboratories to be the pentaglycine interpeptide bridge which is cleaved (Grundling and Schneewind. 2006. J. Bacteriol. 188: 2463-2472; Lu et al. 2006. J. Biol. Chem. 281:549-558). Lysostaphin kills MRSA (Dajcs et al. 2000. Am. J. Ophthalmol. 130: 544), planktonic S. aureus (Walencka et al. 2005. Pol. J. Microbiol. 54:191-200; Wu et al. 2003. Antimicrobial Agents and Chemotherapy 47: 3407-3414), vancomycin-intermediate S. aureus (Patron et al. 1999. Antimicrobial Agents and Chemotherapy 43:1754-1755), and other antibiotic-resistant strains of S. aureus (Bhakta et al. 2003. Indian J. Med. Res. 117:146-151). It is also active against S. aureus biofilms (Walencka et al., supra; Wu et al., supra), and exhibits limited activity against many coagulase negative staphylococci (Cisani et al. 1982. Antimicrobial Agents and Chemotherapy 21:531-535; McCormick et al. 2006b. Curr. Eye Res. 31:225-230; Zygmunt et al. 1968. Appl. Microbiol. 16:1168-1173).
Lysostaphin has a long history of successfully treating livestock and animal models of human disease. Treatment of mouse models with lysostaphin was initiated as early as the 1960s (reviewed in Climo et al. 1998. Antimicrobial Agents and Chemotherapy42:1355-1360). In treating S. aureus bovine mastitis, intramammary infusions did not raise a significant immune response until 18 to 21 injections, while seven once-daily injections were curative (Oldham and Daley. 1991. J. Dairy Sci. 74: 4175-4182). In a S. aureus endocarditis model, systemic lysostaphin treatment yielded minimal adverse effects (Climo et al., supra). Lysostaphin (15 mg/kg) given by i.v. for 9 weeks resulted in serum antibodies to lysostaphin and an eight-fold reduction in its lytic activity, but no adverse immune response (Schaffner et al. 1967. Yale J. Biol. Med. 39: 230-244). More recent studies demonstrate the efficacy of lysostaphins for curing rat nasal colonization (Kokai-Kun et al. 2003. Antimicrobial Agents and Chemotherapy 47: 1589-1597) and rat neonatal S. aureus infections (Oluola et al. 2007. Antimicrobial Agents and Chemotherapy 51:2198-2200). Lysostaphin has also been shown to be effective in a rabbit model of endophthalmitis against coagulase negative staphylococci (McCormick et al. 2006a. Curr. Eye Res. 31:225-230). Lysostaphin expression as a transgenic antimicrobial protects mice and cattle from an intra-mammary challenge by S. aureus (Kerr et al. 2001. Nat. Biotech. 19: 66-70; Wall et a. 2005. Nat. Biotechnol. 23: 445-451). There has been a flood of recent patent applications that take advantage of lysostaphin and phage proteins as antimicrobials (Donovan, D. M. 2007. Recent Patents in Biotechnology 1:113-122).
The use of peptidoglycan hydrolase enzymes as antimicrobials has been tested in combinations with other antimicrobials. Lysostaphin has been shown to be synergistic with β-lactams against oxacillin-resistant S. epidermidis (Kiri et al. 2002. Antimicrobial Agents and Chemotherapy 46:2017-2020), with the catanionic peptide ranalexin against MRSA (Graham and Coote. 2007. J. Antimicrob. Chemother. 59:759-762), and with beta-lactam antibiotics (including benzylpenicillin, methicillin, and cephalosporin B), bacitracin, or polymyxin B, against five clinical S. aureus isolates including MRSA (Polak et al. 1993. Diagn. Microbiol Infect. Dis. 17: 265-270.). Numerous other pathogens were tested for synergy with cationic antimicrobial peptides and lysostaphin. None were affected except S. aureus. Similarly, the phage lytic enzyme Cpl-1 was synergistic with gentamycin, penicillin and with the phage endolysin Pal against several penicillin-resistant and -sensitive S. pneumonia strains (Djurkovic et at 2005. Antimicrobial Agents and Chemotherapy 49:1225-1228.; Loeffler and Fischetti. 2003. Antimicrobial Agents and Chemotherapy 47: 375-377). A recent patent application (Kokai-Kun, J. F. 2003. US 20030211995) indicates there is synergy with lysostaphin and the phi11 endolysin or the antibiotic bacitracin against S. aureus. 
To reduce the use of broad range antibiotics and thus decrease the chance of antibiotic resistance development, our goal is to develop pathogen-specific agents that are effective for the treatment of mastitis and as well as for the treatment of clinical multidrug-resistant bacteria, in particular staphylococci, that have developed resistance to antimicrobial drugs. Methicillin/oxacillin-resistant S. aureus is an example of such multi-drug resistant staphylococci. In this study, we examine some basic properties of the LysK enzyme in order to optimize for its antimicrobial activity and examine the use of His-tagged variants of LysK together with recombinantly produced lysostaphin to demonstrate synergy against the MRSA strain USA300.